Occurrence and characterization of methicillin-resistant Staphylococcus spp. in diseased dogs in Brazil

Staphylococcus pseudintermedius is a major commensal bacterium of the skin and mucosae of dogs and an opportunistic agent responsible for several clinical infections, such as pyoderma, otitis, and surgical wound infections. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has become a problem of great concern in veterinary and human medicine because it is multidrug resistant (MDR) and can also infect humans. This study aimed to identify the occurrence of Staphylococcus spp. in infected patients and investigate the antimicrobial resistance profiles and molecular structure of MRSP isolates. Samples were obtained from two different veterinary clinics; suggestive colonies were submitted to matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry and confirmed at the species level by polymerase chain reaction (PCR). Sequencing of the 16S rRNA and rpoB genes were used in selected samples that were not identified by MALDI-ToF and by the species-specific PCR. Antimicrobial susceptibility and PCR detection of mecA were performed. MRSP isolates were subjected to multilocus sequence typing. Of all the clinical staphylococci (n = 131), 98 (74.8%) were identified as S. pseudintermedius. Multidrug resistance (resistance to ≥3 classes of antimicrobials) was observed in 63.2% of S. pseudintermedius isolates, and 24.5% of S. pseudintermedius isolates were methicillin-resistant. Half of the MRSP isolates were isolated from surgical site infections. Among the ten sequence types (ST) identified, nine were novel. ST71 was the most prevalent and associated with resistance to fluoroquinolones. Prior antimicrobial therapy, hospitalization, and surgical site infections seemed to be risk factors for MRSP acquisition. The present study showed a high rate of MDR staphylococci in infected dogs. MRSP was isolated from different clinical conditions, mainly surgical site infections. Additionally, this is the first study to extensively investigate the population structure of MRSP in Brazil, which revealed the dispersion of CC71 and nine novel ST. These findings raise concerns for both animal and human health due to the zoonotic potential of this species and limited therapeutic options available for MRSP infections.

Introduction Staphylococcus pseudintermedius is a major commensal agent of the skin and mucosae of dogs and integrates the S. intermedius group (SIG) along with three other species: S. intermedius, S. delphini and S. cornubiensis [1,2]. Although it is commonly found in the nose and anus of healthy individuals, S. pseudintermedius can be responsible for several clinical infections, being one of the leading causes of pyoderma, but it is also a common cause of otitis, wound infections, and urinary tract infections [1,[3][4][5].
Over the past decade, the emergence of methicillin-resistant S. pseudintermedius (MRSP) has become a problem of great concern in veterinary medicine [4,6,7] due to the acquisition of the Staphylococcal Cassette Chromosome mec (SCCmec), a mobile genetic element which harbors the mecA gene and confers resistance to all beta-lactam antimicrobials, except for the fifth-generation cephalosporins ceftaroline and ceftobiprole [8]. MRSP strains are also commonly resistant to other antimicrobial classes, such as aminoglycosides, macrolides, and fluoroquinolones, representing a challenge to therapeutic approaches [4,[9][10][11]. Furthermore, SCCmec and/or other antimicrobial resistance genes can be transferred between different staphylococci, which can lead to serious public health issues, especially when involving MRSP and methicillin-resistant S. aureus (MRSA) [9, 12,13]. Similar to MRSP in dogs, MRSA is a major pathogen associated with both hospital and community-acquired infections in humans and is responsible for severe cases of soft tissue infections [11,12].
Reports of human cases of carriage or infection by S. pseudintermedius have been scarcely described over the last few years [9,14]. S. pseudintermedius can cause a range of skin and soft tissue infections in humans, and studies have shown high similarity among strains isolated from human patients and their dogs [15][16][17][18][19]. Furthermore, it seems like long-term contact with dogs may be a risk factor for acquiring the bacterium, with veterinarians and pet owners being the most affected [9,14,20,21]. In this context, MRSP is a relevant one health problem because of its potential zoonotic risk and the limited options of treatment currently available.
In addition to its growing importance, there are few studies on MRSP in Brazil. To date, there is almost no information about its population structure and resistance patterns [22][23][24][25]. Furthermore, few studies have investigated the possible relationship between different clinical manifestations caused by S. pseudintermedius in dogs and their patterns of antimicrobial resistance (AMR) and genetic background. This study aimed to identify the occurrence of staphylococci, with major focus on S. pseudintermedius, in dogs with clinical conditions and investigate its epidemiology in Belo Horizonte, Minas Gerais, by reporting the antimicrobial resistance profiles and analyzing the molecular structure of MRSP isolates. USA) and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI--TOF) mass spectrometry, as described previously [26]. Briefly, for each strain, 1 μl of formic acid (70%) and 1 μl of MALDI-TOF MS matrix, consisting of a saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA) (Bruker Daltonics, Bremen, Germany), were applied to the spot and allowed to air-dry. Spectra were acquired using the FlexControl MicroFlex LT mass spectrometer (Bruker Daltonics) with a 60-Hz nitrogen laser, in which up to 240 laser shots. Parameters for mass range detection were defined to allow the identification from 1,960 to 20,137 m/z, where Ion source 1 v was 19.99 kv, Ion source 2 voltage was 18.24 kv and the lens voltage was 6.0 kv for data acquisition. Prior to measurements, calibration was preceded with a bacterial test standard (E. coli DH5 alpha; Bruker Daltonics). The Real Time (RT) identification score criteria used were those recommended by the manufacturer: score � 2.000 indicates a species-level identification. Isolates identified as SIG by MALDI-ToF were confirmed by multiplex polymerase chain reaction (PCR) of nuc gene [27]. SIG isolates that were not amplified by multiplex PCR, and non-SIG isolates with MALDI-ToF score under 2.000 were submitted to sequencing of the 16S rRNA gene and sequencing of the rpoB gene, if necessary. Amplification and sequencing of the 16S rRNA and rpoB genes were performed as previously described [28,29].

DNA extraction and detection of mecA
Extraction of bacterial genomic DNA with guanidium thiocyanate was performed according to a method previous described [32]. The extracted DNA was quantified using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purity of the extracted DNA was determined using the absorbance ratio at 260/280 nm. All staphylococci isolates were tested by PCR to determine whether they carry the mecA gene [33].

Multilocus sequence typing (MLST)
Multilocus sequence typing was performed for all MRSP isolates, according to literature [34,35]. STs were determined using the S. pseudintermedius MLST database (https://pubmlst.org/ organisms/staphylococcus-pseudintermedius), and new STs were assigned by the curator Vincent Perreten (vincent.perreten@vbi.unibe.ch). Phyloviz v 2.0, using the goeBURST algorithm [36,37], was used to infer the population structure, with clonal complexes (CCs) composed of all strains sharing at least six identical alleles (single-locus variant). Multilocus sequence analysis (MLSA) was performed based on concatenated sequences of the seven alleles from the MLST scheme. A phylogenetic tree was constructed using the approximately-maximum-likelihood (ML) model and Hasegawa-Kishino-Yano models of nucleotide evolution was constructed using IQ-TREE [38], with a bootstrap analysis with 1000 replicates, and visualized and annotated using iTol v.4 [39].

Statistical analysis
Data were summarized using frequency tables and percentages for categorical variables and medians for continuous variables. To measure the association between the categorical variables and isolated strains, a univariate analysis using Fisher's exact test was performed. The associations were expressed as odds ratios (ORs) and their 95% confidence intervals (CIs). Statistical significance was set at P � 0.05. For quantitative variables, the Mann-Whitney test was used, with a p value � 0.05, and Tukey's comparison test was applied if statistical significance was observed. All analyses were performed using R Software 4.0.9 (R Development Core Team, NZ).

Data collection
One hundred and sixteen dogs were included in this study, which provided 131 samples of staphylococcal species. When two isolates from the same animal were obtained from the same site and had the same antimicrobial resistance profile, only one of them was maintained in the study. Most of the isolates (95/131-72.5%) were obtained from HV-UFMG, while 27.5% (36/ 131) samples from 27 animals were obtained in a private clinic. The animals belonged to different breeds and ages ranging from 12 to 204 months, with a mean of 88 months (±46.3). Fifty-seven animals were female, fifty-eight were male, and one was not provided with gender information (see S1 File). At least 64 animals (55.2%) had previous use of antimicrobials, 56 received different systemic antibiotics, eight of them had used only topical antibiotics, and 15 of them used both systemic and topical products. The main prescribed antibiotics were cefalexin (29/64), amoxicillin (9/64), or a combination of amoxicillin and clavulanic acid (16/64) and enrofloxacin (11/64). Most of the clinical specimens were associated with dermatological conditions: 58 (44.3%) with pyoderma, 28 (21.4%) with otitis, 20 (15.3%) with urogenital tract infections, 15 (11.4%) with surgical site infections (SSIs), and 10 (7.6%) from other sites.

Strain isolation and identification
A total of 131 samples were obtained and confirmed to be Staphylococcus spp. by MALDI-ToF. A total of 102 isolates (77.9%) were identified by MALDI-TOF as part of the SIG (Table 1). Among these, PCR of the nuc gene confirmed 98 (96.1%) isolates as S. pseudintermedius and four (3.9%) as S. delphini. Sequencing of the 16S rRNA gene confirmed all species, except in one case. The only sample sent for sequencing of the rpoB gene was identified as S. delphini, confirming the MALDI-ToF results. The most frequent species isolated was S. pseudintermedius (98/131, 74.8%), followed by S. schleiferi (20/131, 15.3%) (see Fig 1), although it is important to state that the possible differentiation of S. schleiferi into two species, S. coagulans and S. schleiferi [40], was not investigated. Nine animals had both pyoderma and otitis at time of sampling-two of them caused by S. pseudintermedius and two caused by S. schleiferi; in all those cases, isolates from the same animal showed the same antimicrobial resistance profile. The other five animals had otitis and pyoderma caused by different species, more specifically a combination of S. pseudintermedius and S. schleiferi in four of them and the other one with a combination of S. pseudintermedius and S. delphini. Two dogs with pyoderma carried methicillin-susceptible S. pseudintermedius (MSSP) with different patterns of antimicrobial resistance, one carried both MSSP and MRSP and two carried S. pseudintermedius and S. aureus.

Methicillin-resistant staphylococci
Approximately 23.5% (24/102) of SIG isolates were positive for the mecA gene, all of them identified as S. pseudintermedius. Three (10.3%) of non-SIG isolates were also positive for mecA. Of these, two were S. schleiferi and one was S. epidermidis. However, only S. epidermidis was phenotypically resistant to oxacillin or cefoxitin (inhibition halo = 24mm for FOX); this strain was obtained from a dog with a nosocomial UTI. The two S. schleiferi positive for mecA were not resistant to oxacillin (inhibition halos for FOX 19 and 19,1mm). No MRSA isolates were identified in this study.

Predictors for MRSP
There was no significant difference in the proportion of MRSP isolates between female and male dogs or between age groups. However, previous antimicrobial treatment (OR9.21 95%CI 2.05-41.25) and hospital-acquired infections were associated with the isolation of MRSP strains. When considering the clinical condition presented by the animals, SSIs were

Discussion
In the present study, we characterized the occurrence of different species of Staphylococcus spp. isolated from several different tissues in dogs and evaluated their antibiotic resistance. S. pseudintermedius was the most prevalent species in this study. As it is the major causative agent of pyoderma in dogs, most clinical specimens were obtained from skin lesions [1,41]. The second most frequently observed species was S. schleiferi, but it is important to remember that the recently approved differentiation of S. schleiferi into S. coagulans and S. schleiferi [40] was not considered in the present work. Anyway, S. schleiferi is rarely found in healthy carriers and is commonly isolated from dogs with otitis or pyoderma [11,42,43]. Some animals were infected by both MSSP and MRSP, or even by two different species of Staphylococcus spp., similar to previous reports [1,11]. As these bacteria can share MGE, it is worrisome that strains previously susceptible could acquire resistance and/or virulence genes when in contact with virulent/resistant lineages [10,12].
High rates of AMR were observed among the SIG isolates to most of the antimicrobials tested in this study. AMR is considered a natural phenomenon, but the excessive or inappropriate use of antimicrobial drugs in human and veterinary medicine accelerates its occurrence [44]. Over half of the animals included in the study had been previously treated with at least one systemic antibiotic, which can be linked to the high rates of AMR found. Unfortunately,  (2) Skin (1) OXA, PEN, ERY, CLI, SXT, RIF, CIP, GEN Surgical wound (1) Skin (1) Others (  this frequency is not surprising, considering the main clinical manifestations presented by those animals. In small animal practice, skin infections are commonly seen in routine, being the main reason for antimicrobial administration in dogs, accounting for almost one-third of antimicrobial prescriptions in small animal clinics [45]. Similarly, otitis and bacterial urinary tract disease are common causes of morbidity in dogs and are among the leading causes of antimicrobial use [46][47][48]; and surgery often requires antimicrobial prophylaxis and postoperative administration of antimicrobials [49,50]. The main prescribed antibiotics were cefalexin, amoxicillin, amoxicillin/clavulanic acid, and enrofloxacin. Beta-lactams are commonly the most used antimicrobial group in small animals, especially when culture is not available [12,14,47]. Except for penicillinase-labile antibiotics, MSSP usually presents satisfactory rates of antimicrobial susceptibility reported in the literature [10,[51][52][53]. However, our results revealed high rates of antimicrobial resistance to several classes of antibiotics, including veterinary critically important antimicrobial agents [54], in both MSSP and MRSP strains, with significant occurrence of MDR. In contrast, chloramphenicol and nitrofurantoin were the antibiotics with major rates of susceptibility among S. pseudintermedius strains, which is expected, since both are forbidden for veterinary use in Brazil [55].
The present study identified an overall occurrence of 24.5% of MRSP among all S. pseudintermedius isolates, which is within the expected range, since literature reports variable rates of prevalence of MRSP in dogs, ranging from 0% to 60% [3,9,56]. Previous studies conducted in Brazil showed similar or higher rates of MRSP in animals with pyoderma, otitis and UTI [22][23][24][25]46] when compared to the present work. It is important to emphasize that comparisons between different studies are not easy to make, as the prevalence of MRSP depends not only on the geographic region but also on the type of sample, the population included, and methodology of the study [1,57].
Interestingly, over 90% of S. pseudintermedius strains isolated from surgical wounds were MRSP. Statistical analysis revealed that SSIs, prior antimicrobial treatment, and nosocomial infections are associated with the occurrence of MRSP. This is in agreement with the literature, which has demonstrated that prior antimicrobial exposure, hospitalization, and skin lesions and/or surgical wounds are associated with a higher risk of acquiring MRSP [9, 35,[58][59][60]. Furthermore, it is possible that the large use of amoxicillin/clavulanic acid, cephalosporins, and fluoroquinolones, which were the main antimicrobials prescribed to dogs included in this study, may be linked to the selection of MRSP [12,14].
Previous studies using MLST provided information about the population structure of MRSP and have shown that dissemination of this pathogen is linked to some successful clonal lineages, particularly CC71 and CC258 in Europe, CC68 in North America, and both CC45 and CC112 in Asia [56,61,62]. In Brazil, only one study has reported the CC of their isolatesa single MRSP ST71 strain isolated from the nostril of a healthy dog [63], so there is no extensive knowledge about the genetic background of S. pseudintermedius currently circulating in the country. In the present study, approximately 41.7% (10/24) of the MRSP strains belonged to CC71, which was also reported in Brazil previously [63]. Studies have shown that CC71 is frequently resistant to all antimicrobials used in the routine of small animals, including macrolides, aminoglycosides, and fluoroquinolones, and that it can be associated with higher rates of resistance to oxacillin, amoxicillin/clavulanic acid, cephalothin, and ampicillin [4,21,64]. In fact, isolates belonging to CC71 in the present study were all resistant to at least six classes of non-beta-lactam antimicrobials, including fluoroquinolones, which are associated with the successful spread of MDR MRSP lineages [53]. Most CC71 strains were isolated from SSIs at the same veterinary hospital (data not shown), which raised the concern of possible nosocomial transmission as previously reported [59]. Further molecular characterization is required to confirm this hypothesis.
Recent studies across Europe, North America, and Oceania have shown that the prevalence of new lineages is increasing, possibly overcoming the prevalence of CC71 within years [4,5,35,62,65]. In the present study, 62.5% (15/24) of the MRSP strains belonged to nine new STs. All STs were resistant to at least three different classes of non-beta-lactam antimicrobials, and some of them were isolated from more than one animal. This observation corroborates the hypothesis that events of horizontal gene transfer may be as relevant as clonal dispersion to MRSP and highlights the need for more studies in Brazil to better understand the evolution of these new lineages [1,4,35,56]. This is the first study to extensively investigate the phenotypic and molecular characteristics of S. pseudintermedius isolates in Brazil. In addition, this study identified risk factors for acquisition of MRSP and analyzed the population structure of MRSP strains, which contributes to understanding the dispersion of MRSP not only in Brazil, but also in South America, where there is still a lack of information about it. The present study confirmed that S. pseudintermedius is the main staphylococcal species isolated from infected dogs in Belo Horizonte. The high rate of MDR observed, especially MRSP-MDR strains, is of great concern. CC71 is currently circulating among Brazilian veterinary clinics and poses a challenge to therapeutic protocols due to its resistance to most antimicrobial classes of daily use in small animal practice and its rapid dispersion. Nevertheless, the new STs found in this study indicate that the spread of MRSP via horizontal gene transfer may be as relevant as clonal dispersion. To our knowledge, this is the first Brazilian report that identified MRSP ST71 in diseased dogs and revealed new circulating STs in the country.